scholarly journals A Wi-Fi live streaming Centrifuge Force Microscope for benchtop single-molecule experiments

2020 ◽  
Author(s):  
Jibin Abraham Punnoose ◽  
Andrew Hayden ◽  
Lifeng Zhou ◽  
Ken Halvorsen
Keyword(s):  
High Throughput ◽  
Single Molecule ◽  
Base Pair ◽  
Ease Of Use ◽  
Live Streaming ◽  
Dna Duplexes ◽  
Kinetic Measurements ◽  
Dissociation Kinetic ◽  
The Difference ◽  
Motor Operation

AbstractThe ability to apply controlled forces to individual molecules has been revolutionary in shaping our understanding of biophysics in areas as diverse as dynamic bond strength, biological motor operation, and DNA replication. However, the methodology to perform single-molecule experiments has been and remains relatively inaccessible due to cost and complexity. In 2010, we introduced the Centrifuge Force Microscope (CFM) as a new platform for accessible and high-throughput single-molecule experimentation. The CFM consists of a rotating microscope where prescribed centrifugal forces can be applied to microsphere-tethered biomolecules. In this work, we develop and demonstrate a next-generation Wi-Fi CFM that offers unprecedented ease of use and flexibility in design. The modular CFM unit fits within a standard benchtop centrifuge and connects by Wi-Fi to a external computer for live control and streaming at near gigabit speeds. The use of commercial wireless hardware allows for flexibility in programming and provides a streamlined upgrade path as Wi-Fi technology improves. To facilitate ease of use, detailed build and setup instructions are provided, as well as LabVIEW™ based control software and MATLAB® based analysis software. We demonstrate the analysis of force-dependent dissociation of short DNA duplexes of 7, 8, and 9 bp using the instrument. We showcase the sensitivity of the approach by resolving distinct dissociation kinetic rates for a 7 bp duplex where one G-C base pair is mutated to an A-T base pair.SignificanceThe ability to apply mechanical forces to individual molecules has provided unprecedented insight into many areas of biology. Centrifugal force provides a way to increase the throughput and to decrease the cost and complexity of single-molecule experiments compared to other approaches. In this work, we develop and demonstrate a new user-friendly Centrifuge Force Microscope (CFM) that enables live-streaming of high-throughput single-molecule experiments in a benchtop centrifuge. We achieved near gigabit bandwidth with standard Wi-Fi components, and we provide detailed design instructions and software to facilitate use by other labs. We demonstrate the instrument for sensitive kinetic measurements that are capable of resolving the difference between two DNA duplexes that differ by a single G-C to A-T substitution.

scholarly journals Single-molecule assay for proteolytic susceptibility using centrifuge force microscopy:force-induced destabilization of collagen‘s triple helix

2017 ◽  
Author(s):  
Michael W.H. Kirkness ◽  
Nancy R. Forde
Keyword(s):  
Real Time ◽  
High Throughput ◽  
Single Molecule ◽  
Triple Helix ◽  
Proteolytic Cleavage ◽  
Ease Of Use ◽  
Enzymatic Assay ◽  
Biomolecular Structure ◽  
Collagen Molecules ◽  
Time Readout

Force plays a key role in regulating dynamics of biomolecular structure and interactions, yet techniques are lacking to manipulate and continuously read out this response with high throughput. We present an enzymatic assay for force-dependent accessibility of structure that makes use of a wireless mini-radio centrifuge force microscope (MR.CFM) to provide a real-time readout of kinetics. The microscope is designed for ease of use, fits in a standard centrifuge bucket, and offers high-throughput, video-rate readout of individual proteolytic cleavage events. Proteolysis measurements on thousands of tethered collagen molecules show a load-enhanced trypsin sensitivity, indicating destabilization of the triple helix.

scholarly journals A wireless centrifuge force microscope (CFM) enables multiplexed single-molecule experiments in a commercial centrifuge

2016 ◽  
Author(s):  
Tony Hoang ◽  
Dhruv S. Patel ◽  
Ken Halvorsen
Keyword(s):  
Single Molecule ◽  
Low Cost ◽  
Ease Of Use ◽  
Wireless Transmission ◽  
Massively Parallel ◽  
Dna Duplexes ◽  
Research Grade ◽  
Force Clamp ◽  
Transmission Capability ◽  
Improve Access

AbstractThe centrifuge force microscope (CFM) was recently introduced as a platform for massively parallel single-molecule manipulation and analysis. Here we developed a low-cost and selfcontained CFM module that works directly within a commercial centrifuge, greatly improving accessibility and ease of use. Our instrument incorporates research grade video microscopy, a power source, a computer, and wireless transmission capability to simultaneously monitor many individually tethered microspheres. We validated the instrument by performing single-molecule force shearing of short DNA duplexes. For a 7 bp duplex, we collected over 1000 statistics of force dependent shearing kinetics from 2 pN to 12 pN with dissociation times in the range of 10-100 seconds. We extended the measurement to a 10 bp duplex, applying a 12 pN force clamp and directly observing single-molecule dissociation over an 85 minute experiment. Our new CFM module facilitates simple and inexpensive experiments that dramatically improve access to singlemolecule analysis.

scholarly journals Hidden Hoogsteens in the Data

2014 ◽  
Vol 70 (a1) ◽  
pp. C1508-C1508
Author(s):  
Bradley Hintze ◽  
Huiqing Zhou ◽  
Hashim Al-Hashimi ◽  
David Richardson ◽  
Jane Richardson
Keyword(s):  
Electron Density ◽  
High Throughput ◽  
Base Pair ◽  
Base Pairing ◽  
Base Pairs ◽  
X Ray ◽  
Difference Density ◽  
High Throughput Method ◽  
Dna Mismatches ◽  
The Difference

Although extremely rare in the crystallographic database, Hoogsteen (HG) base pairs have been modeled, especially at duplex termini, protein-DNA interfaces, damaged DNA and DNA mismatches. Formation of HG base pairs in duplex DNA requires a 180 degree rotation along the glycosidic bond of the purine relative to the canonical Watson-Crick (WC) base-pair. In a recent survey of the PDB we identified 75 HS base pairs in 2,910 x-ray derived models. Recent evidence suggests that the prevalence of HG base pairs in nature to be greater than previously thought. Due to electron density density ambiguity at mid to low resolution, it is possible for bases to be modeled incorrectly. We have developed a high-throughput method which examines the difference density around DNA to identify mis-modeled bases. Here we show where bases were incorrectly modeled and our subsequent fixes which show pairs modeled as WC that really are HG, vice versa, and evidence of WC-HG alternates. This research shows that base pairing conformations in DNA are more heterogeneous than previously thought.

scholarly journals The more the merrier: high-throughput single-molecule techniques

2017 ◽  
Vol 45 (3) ◽  
pp. 759-769 ◽  
Author(s):  
Flynn R. Hill ◽  
Enrico Monachino ◽  
Antoine M. van Oijen
Keyword(s):  
High Throughput ◽  
Single Molecule ◽  
Molecular Mechanisms ◽  
Zero Mode ◽  
Magnetic Tweezers ◽  
Ease Of Use ◽  
Single Molecule Fluorescence ◽  
Significant Challenge ◽  
Large Numbers ◽  
Very High

The single-molecule approach seeks to understand molecular mechanisms by observing biomolecular processes at the level of individual molecules. These methods have led to a developing understanding that for many processes, a diversity of behaviours will be observed, representing a multitude of pathways. This realisation necessitates that an adequate number of observations are recorded to fully characterise this diversity. The requirement for large numbers of observations to adequately sample distributions, subpopulations, and rare events presents a significant challenge for single-molecule techniques, which by their nature do not typically provide very high throughput. This review will discuss many developing techniques which address this issue by combining nanolithographic approaches, such as zero-mode waveguides and DNA curtains, with single-molecule fluorescence microscopy, and by drastically increasing throughput of force-based approaches such as magnetic tweezers and laminar-flow techniques. These methods not only allow the collection of large volumes of single-molecule data in single experiments, but have also made improvements to ease-of-use, accessibility, and automation of data analysis.

Highly Sensitive Detection of Paraquat with Pillar[5]arene as an aptamer in α-Hemolysin Nanopore

2021 ◽  
Author(s):  
Xiaojia Jiang ◽  
Mingsong Zang ◽  
Fei Li ◽  
Chunxi Hou ◽  
Quan Luo ◽  
...  
Keyword(s):  
Real Time ◽  
High Throughput ◽  
Single Molecule ◽  
Single Molecule Detection ◽  
Sensitive Detection ◽  
High Throughput Analysis ◽  
Throughput Analysis ◽  
The Real ◽  
Highly Sensitive ◽  
Highly Sensitive Detection

Biological nanopore-based techniques have attracted more and more attention recently in the field of single-molecule detection, because they allow the real-time, sensitive, high-throughput analysis. Herein, we report an engineered biological...

scholarly journals High throughput transepithelial electrical resistance (TEER) measurements on perfused membrane-free epithelia

Lab on a Chip ◽  
2021 ◽  
Author(s):  
A. Nicolas ◽  
F. Schavemaker ◽  
K. Kosim ◽  
D. Kurek ◽  
M. Haarmans ◽  
...  
Keyword(s):  
High Throughput ◽  
Electrical Resistance ◽  
Drug Exposure ◽  
Transepithelial Electrical Resistance ◽  
Ease Of Use ◽  
Inflammatory Processes

We present an instrument for simultaneously measuring TEER in up to 80 perfused epithelial tubules on an OrganoPlate. The sensitivity, speed and ease of use enables screening of tubules during formation, drug exposure and inflammatory processes.

scholarly journals Tuning Single-Molecule Conductance by Controlled Electric Field-Induced trans-to-cis Isomerisation

2021 ◽  
Vol 11 (8) ◽  
pp. 3317
Author(s):  
C.S. Quintans ◽  
Denis Andrienko ◽  
Katrin F. Domke ◽  
Daniel Aravena ◽  
Sangho Koo ◽  
...  
Keyword(s):  
Electric Field ◽  
Electric Fields ◽  
Single Molecule ◽  
Quantum Interference ◽  
Single Molecule Level ◽  
Wet Chemical Synthesis ◽  
Single Molecule Junctions ◽  
Tunnelling Microscopy ◽  
The Difference

External electric fields (EEFs) have proven to be very efficient in catalysing chemical reactions, even those inaccessible via wet-chemical synthesis. At the single-molecule level, oriented EEFs have been successfully used to promote in situ single-molecule reactions in the absence of chemical catalysts. Here, we elucidate the effect of an EEFs on the structure and conductance of a molecular junction. Employing scanning tunnelling microscopy break junction (STM-BJ) experiments, we form and electrically characterize single-molecule junctions of two tetramethyl carotene isomers. Two discrete conductance signatures show up more prominently at low and high applied voltages which are univocally ascribed to the trans and cis isomers of the carotenoid, respectively. The difference in conductance between both cis-/trans- isomers is in concordance with previous predictions considering π-quantum interference due to the presence of a single gauche defect in the trans isomer. Electronic structure calculations suggest that the electric field polarizes the molecule and mixes the excited states. The mixed states have a (spectroscopically) allowed transition and, therefore, can both promote the cis-isomerization of the molecule and participate in electron transport. Our work opens new routes for the in situ control of isomerisation reactions in single-molecule contacts.

scholarly journals Parallel, linear, and subnanometric 3D tracking of microparticles with Stereo Darkfield Interferometry

2021 ◽  
Vol 7 (6) ◽  
pp. eabe3902
Author(s):  
Martin Rieu ◽  
Thibault Vieille ◽  
Gaël Radou ◽  
Raphaël Jeanneret ◽  
Nadia Ruiz-Gutierrez ◽  
...  
Keyword(s):  
High Precision ◽  
High Throughput ◽  
Single Molecule ◽  
Particle Tracking ◽  
Focal Plane ◽  
Tracking System ◽  
Three Dimensional ◽  
3D Tracking ◽  
Single Molecule Force Spectroscopy ◽  
A New Technique

While crucial for force spectroscopists and microbiologists, three-dimensional (3D) particle tracking suffers from either poor precision, complex calibration, or the need of expensive hardware, preventing its massive adoption. We introduce a new technique, based on a simple piece of cardboard inserted in the objective focal plane, that enables simple 3D tracking of dilute microparticles while offering subnanometer frame-to-frame precision in all directions. Its linearity alleviates calibration procedures, while the interferometric pattern enhances precision. We illustrate its utility in single-molecule force spectroscopy and single-algae motility analysis. As with any technique based on back focal plane engineering, it may be directly embedded in a commercial objective, providing a means to convert any preexisting optical setup in a 3D tracking system. Thanks to its precision, its simplicity, and its versatility, we envision that the technique has the potential to enhance the spreading of high-precision and high-throughput 3D tracking.

scholarly journals Single molecule high-throughput footprinting of small and large DNA ligands

2017 ◽  
Vol 8 (1) ◽  
Author(s):  
Maria Manosas ◽  
Joan Camunas-Soler ◽  
Vincent Croquette ◽  
Felix Ritort
Keyword(s):  
High Throughput ◽  
Single Molecule

scholarly journals Nearfield trapping increases lifetime of single-molecule junction by one order of magnitude

2020 ◽  
Author(s):  
Albert C. Aragonès ◽  
Katrin F. Domke
Keyword(s):  
Molecular Electronics ◽  
Single Molecule ◽  
Fold Increase ◽  
Trapping Efficiency ◽  
Non Invasive ◽  
Order Of Magnitude ◽  
Molecule Junction ◽  
Single Molecule Junction ◽  
The Difference ◽  
Power Densities

Abstract Progress in molecular electronics (ME) is largely based on improved understanding of the properties of single molecules (SM) trapped for seconds or longer to enable their detailed characterization. We present a plasmon-supported break-junction (PBJ) platform to significantly increase the lifetime of SM junctions of 1,4-benzendithiol (BDT) without the need for chemical modification of molecule or electrode. Moderate far-field power densities of ca. 11 mW/µm2 lead to a >10-fold increase in minimum lifetime compared to laser-OFF conditions. The nearfield trapping efficiency is twice as large for bridge-site contact compared to hollow-site geometry, which can be attributed to the difference in polarizability. Current measurements and tip-enhanced Raman spectra confirm that native structure and contact geometry of BDT are preserved during the PBJ experiment. By providing a non-invasive pathway to increase short lifetimes of SM junctions, PBJ is a valuable approach for ME, paving the way for improved SM sensing and recognition platforms.

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